Energy and nutrient homeostasis is maintained through a complex regulatory network formed by signaling and transcriptional components that control metabolic genes. In this regulatory network, metabolic flexibility in skeletal muscle is defined as the ability to undergo a nutrient switch of fuel substrate utilization between glucose and fatty acids. Clinically, the metabolic syndrome that include an array of risk factors such insulin resistance, obesity and dyslipidemia among others is associated with a loss of metabolic flexibility. Moreover, it is though that it is this lack of metabolic flexibility that correlates with an incomplete mitochondrial oxidation of fatty acids and increased accumulation of intramyocellular lipids, a putative cause of insulin resistance. We have identified an important component of the low nutrient/fasting switch from glucose to fatty acid oxidation that involves the SIRT1 deacetylase enzyme that targeting and activating the transcriptional coactivator PGC- 1a promotes this nutrient switch. Mimicking fasting metabolic response, active and deacetylated PGC-1a increases expression of genes encoding for regulatory proteins and enzymes linked to mitochondrial fatty acid oxidation. Interestingly, this pathway is altered in skeletal muscle of mice fed with high fat diet in which PGC- 1a is highly acetylated and correlates with a loss of metabolic flexibility and insulin resistance. Although activation of SIRT1 and PGC-1a are key regulators of this process, how low nutrient signals control SIRT1 enzymatic activity and how mechanistically deacetylated PGC-1a is a hyperactive protein is unknown. The major goal of this proposal is to identify the mechanisms by which fasting and low nutrient signals to SIRT1 enzymatic activity to induce PGC-1a deacetylation in skeletal muscle and to investigate their metabolic functionality in in-vivo mouse models. We have three aims: Aim 1 is to perform molecular and functional analysis of how fasting/low glucose controls SIRT1 deacetylase enzymatic activity in in-vitro as well in-vivo conditions. Aim 2 is to carry out molecular and functional analysis of PGC-1a acetylation and identify new proteins that account for the hyperactivity of deacetylated PGC-1. Aim 3 will determine the effects of fasting/PKA activation on glucose and lipid metabolism through generation of skeletal muscle transgenic mice expressing SIRT1 and PGC-1a mutant alleles in different dietary conditions. This investigation will allow us to identify the molecular mechanisms by which fasting and PKA activation in skeletal muscle controls glucose and lipid metabolism. Since dysregulation of these processes that involves a loss of metabolic flexibility is a major component of the metabolic syndrome including obesity and diabetes, our studies on the regulation of SIRT1 deacetylase activities and PGC-1a acetylation might translate into potential therapeutic interventions. PUBLIC HEALTH RELEVANCE: Dysregulation of the SIRT1/PGC-1a function in skeletal muscle leads to loss of metabolic flexibility and altered mitochondrial oxidation. Since this is a hallmark of metabolic diseases such as obesity and type 2 diabetes, studies in this grant proposal might translate into potential therapies for these diseases.